Methods for removing suspended particles from soluble protein solutions

ABSTRACT

The present invention provides soluble protein solutions, free of suspended particles in high yield. More particularly, the current invention provides a method for removing suspended particles from soluble protein solutions by filtering the soluble protein solution through highly purified diatomaceous earth.

This application claims the benefit under 35 U.S.C. § 119 (e) of U.S.Provisional Application No. 60/241,967, filed Oct. 19, 2000, which isincorporated herein by reference in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to methods for removing suspendedparticles from soluble protein solutions. In particular, the methods ofthe invention are useful for removing suspended particles from secretedprotein solutions and lysates, including bacterial lysates containing aheterologous protein.

2. BACKGROUND OF THE INVENTION

Proteins play critical roles in functions such as metabolism, geneexpression, signal transduction, cellular and extracellular structures,which are essential to the survival and/or reproduction of any livingorganism. Many proteins may be used in therapeutic and/or diagnosticapplications, particularly when available in pure form. Contaminantsoften prevent realization of therapeutic and/or diagnostic goals and mayendanger the health of a patient.

Protein purification is often a significant challenge, especially whenlarge amounts of protein are required for therapeutic or diagnosticpurposes. Procedures that simply and rapidly provide the protein ofinterest in pure form and high yield are very desirable, regardless ofscale.

Removing suspended particles from soluble protein solutions is often animportant practical problem in purifying proteins of therapeutic ordiagnostic significance, particularly when heterologous proteins areexpressed in either eukaryotic or procaryotic cells. Currently, severalmethods are used for removing suspended particles from soluble proteinsolutions.

Centrifugation is a common method for removing suspended particles fromsoluble protein solutions. In some situations, suspended particles maybe removed from soluble protein solutions by centrifugation alone. Inother instances, prior to centrifugation, soluble protein solutions,particularly bacterial lysates, may be treated with a flocculating agent(e.g., polyethyleneimine (“PEI”)) which typically removes macromolecules(e.g., DNA and endotoxins) and cell debris. However, large scalecentrifugation equipment is very expensive capital equipment and isoften a limiting factor in removing suspended particles from solubleprotein solutions on a process scale. Major problems with centrifugationinclude low yields and air entrapment in the supernatant that can leadto substantial protein denaturation. Typical yields of protein aftercentrifugation are about 80%-85%.

Aqueous two-phase partitioning is another method that has been used forremoving cellular debris and suspended particles from soluble proteinsolutions. Liquid-liquid extraction relies on the incompatibilitybetween two polymers in aqueous solution or one polymer and a saltpresent at high concentration. This incompatibility typically results inthe formation of two separate phases of very different compositions. Theprotein molecules partition preferentially into one phase or the other,depending on their characteristics (Hayenga et al., U.S. patentapplication Ser. No. 09/307,549; Diamond et al., Advances in Biochem.Eng. Biotechn. 1992, 47:89-135).

However, aqueous two-phase extraction is time consuming, expensive andrequires large amounts of chemicals, which must be properly disposed incompliance with environmental regulations. Further, the chemicals usedin extraction must be removed from the protein of interest and thetwo-phase distribution of protein may limit product yield. Finally,two-phase extraction lacks generality since only a limited number ofproteins can be purified by this method.

Microfiltration is another popular method for removing suspendedparticles from soluble protein solutions. Microfiltration uses membranesthat either entrap particles on the membrane surface or within a bed offibers found within the membrane. However, microfiltration on a processscale is a complicated operation that requires precise optimization of anumber of variables such as transmembrane pressure, shear force, flowrate, concentration, pH, ionic strength, etc. Thus, process scalemicrofiltration frequently requires considerable development time.

Accordingly, what is needed is a rapid and inexpensive process thatremoves suspended particles from soluble protein solutions in highyield, particularly on a process scale. Further, such a process shouldnot require the use of expensive capital equipment or large amounts ofchemicals that require costly disposal.

3. SUMMARY OF THE INVENTION

The present invention addresses this need by providing rapid, efficientand inexpensive methods for removing suspended particles from solubleprotein solutions. The present invention provides soluble proteinsolutions, free of suspended particles in high yield, while avoiding theuse of expensive capital equipment or chemicals that require expensivedisposal.

The current invention provides a method for removing suspended particlesfrom soluble protein solutions by filtering the soluble protein solutionthrough highly purified diatomaceous earth. Preferably, the highlypurified diatomaceous earth is Celpure™ P-1000.

In one embodiment, the soluble protein solution is a secreted proteinsolution. In another embodiment, the soluble protein solution is alysate. In a preferred embodiment, the lysate is a bacterial lysate.

Preferably, the amount of DNA and endotoxins in a bacterial lysate isreduced. Then, the lysate is filtered through highly purifieddiatomaceous earth to remove suspended particles, which dramaticallyreduces lysate turbidity. In one embodiment, the highly purifieddiatomaceous earth is packed in a filter press.

In a preferred embodiment, flocculation with polyethyleneimine atbetween about pH 7.3 and about pH 7.7 reduces the amount of DNA andendotoxins in the lysate. Preferably, the amount of DNA in the lysate isreduced by between about 100-fold and about 150-fold. In one embodiment,the amount of endotoxins in the lysate is reduced by between about1,000-fold and about 10,000-fold. In another embodiment, the turbidityof the lysate is reduced by between about 200-fold and about 300-fold.

In another preferred embodiment, the lysate is filtered through highlypurified diatomaceous earth with a filter press. In a more specificembodiment, the lysate is stirred with highly purified diatomaceousearth before filtering through the filter press. Preferably, the yieldof the soluble protein solution is between about 95% and about 100%after filtration through highly purified diatomaceous earth.

In yet another preferred embodiment, the lysate is a bacterial lysatecontaining a heterologous protein that was obtained by expression inbacteria. Preferably, the heterologous protein is SY161, which has theamino acid sequence shown in SEQ. ID. NO. 1. In a more specificembodiment, refractile bodies in the lysate are resolubilized.Preferably, the bacteria is E. coli.

In one embodiment, the cysteine residues of the heterologous protein areblocked. Preferably, the cysteine residues are blocked with an oxidizingagent. More preferably, the oxidizing agent is a mixture of sodiumsulfite and sodium tetrathionate. Even more preferably, about a 2:1ratio of sodium sulfite and sodium tetrathionate are wadded to theheterologous protein at a pH of between about 7.8 and about 8.2.

In another embodiment, the blocked cysteine residues of the heterologousprotein are deblocked. Preferably, a reducing agent is used to deblockthe heterologous protein. More preferably, the reducing agent isdithiothreitol.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the amino acid sequence (SEQ ID NO 1) of SY161.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for removing suspendedparticles from soluble protein solutions. The details for practicing theinvention are described in the subsections below.

5.1 Sources of Soluble Protein Solutions

Soluble protein solutions may be prepared by any art-known technique.Thus, for example, soluble protein solutions may be obtained byculturing procaryotes that secrete either wild-type or heterologousproteins, lysis of procaryotes, lysis of procaryotes that expressheterologous proteins, lysis of eucaryotes, lysis of eucaryotesexpressing heterologous proteins, growing eucaryotes that secrete asoluble protein, dissolving commercially available proteins in solution,etc.

Procaryotes can provide soluble protein solutions after cell lysis.Alternatively, microorganisms that secrete either wild type orheterologous proteins may be cultured to provide soluble proteinsolutions. Wild-type prokaryotic cells or those expressing heterologousproteins, can be grown under a variety of conditions known to theskilled artisan. Methods of growing inocula and inoculating culturingmedium are known to the skilled artisan and exemplary methods have beendescribed in the art. Preferred media, times, temperatures and pH forculturing microorganisms are also known in the art. Thus, for example,the cells are grown in a medium suitable for growth of such cells, forexample, minimal media or complete (i.e., rich) media.

Soluble protein solutions containing a heterologous protein may beadvantageously produced by recombinant DNA technology using techniqueswell known in the art for expressing genes. These methods include, forexample, in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination. See, for example, the techniquesdescribed in Sambrook et al., “Molecular Cloning,” Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Vols. 1-3: (1989), andperiodic updates thereof, and Ausubel et al., eds., 1989, “CurrentProtocols in Molecular Biology,” Green Publishing Associates, Inc., andJohn Wiley & Sons, Inc., New York. DNA and RNA encoding any heterologousprotein may be chemically synthesized using, for example, synthesizers.See, for example, the techniques described in “OligonucleotideSynthesis”, 1984, Gait, M. J. ed., GIRL Press, Oxford.

A variety of host-expression vector systems may be utilized to expressproteins. The expression systems that may be used for purposes of theinvention are microorganisms such as bacteria (e.g., E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, phasmid DNA orcosmic DNA expression vectors containing a nucleotide sequence encodingthe desired protein; yeast (e.g., Saccharomyces, Pichia) transfectedwith recombinant yeast expression vectors containing a nucleotidesequence encoding the protein of interest; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containinga nucleotide sequence encoding the protein of interest; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransfected with recombinant plasmid expression vectors (e.g., Tiplasmid) containing a nucleotide sequence encoding the protein ofinterest; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3,U937) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter).

In eukaryotic systems, a number of selection systems may be used, suchas for example, the herpes simplex virus thymidine kinase (Wigler etal., 1977, Cell, 11, 223), hypoxanthine-guaninephosphoribosyltransferase (Szybalska et al., 1962, Proc. Natl. Acad.Sci., USA 48, 2026), and adenine phosphoribosyltransferase (Lowy et al.,1980, Cell, 22, 817) genes can be employed in tk⁻, hprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci., USA 77, 3567;O'Hare et al., 1981, Proc. Natl. Acad. Sci., USA 78, 1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan et al., 1981, Proc.Natl. Acad. Sci. USA 78, 2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol. 150,1); and hygro, which confers resistance to hygromycin (Santerre et al.,1984, Gene, 30, 147).

In bacterial systems, as previously mentioned, a number of expressionvectors may be selected. Bacteria suitable for the practice of theinvention are gram positive and gram negative bacteria. In a preferredembodiment, soluble protein solutions are obtained by expression ofheterologous proteins in Eschericia coli(“E. coli”) and subsequent celllysis. The protein can be expressed in a procaryotic cell usingexpression systems known to those of skill in the art of biotechnology.Expression systems useful for the practice of the current invention aredescribed in U.S. Pat. Nos. 5,795,745; 5,714,346; 5,637,495; 5,496,713;5,334,531; 4,634,677; 4,604,359; 4,601,980, all of which areincorporated herein by reference in their entirety.

Procaryotic cells can be grown under a variety of conditions known tothe skilled artisan. In one aspect of the current invention, the cellsare grown in a medium suitable for growth of such cells, for example,minimal media or complete (i.e., rich) media.

Staphylokinase is a potent, uniquely fibrin-selective thrombolytic agentthat has substantial therapeutic value in the treatment of acutemyocardial infarction and ischemic stroke (Collen, Nature Medicine,1998, 279). The staphylokinase gene has been cloned from a variety ofsources including a Staphylococcus aureus strain and has been expressedin E. coli (Sako et al., Mol. Gen. Genet., 1983, 271; Behnke et al.,Mol. Gen. Genet., 1987, 528; Colleen et al., Fibrinolysis 6, 203). Anumber of natural variants of the staphylokinase gene fromStaphylococcus aureus have been isolated (Kim et al., ThrombosisResearch, 1997, 387).

SY161 is a staphylokinase analogue that differs at thirteen amino acidsfrom the amino acid sequence of the native protein, while retainingsignificant in vivo thrombolytic activity (Collen et al., Circulation102, 1766, 2000). A modification of SY161 (i.e., attachment ofpolyethylene glycol to the lone cysteine in the protein) that increaseshalf-life and decreases immunogenicity is currently in clinical trialsfor treatment of myocardial infarction and peripheral arterialocclusion. A preferred soluble protein solution that may be used inpracticing the current invention may be obtained by lysis of E. colistrains that express SY161.

5.2. Removing Suspended Particles from Soluble Protein Solutions

The current invention removes suspended particles from soluble proteinsolutions. Soluble protein solutions may be obtained from cells, cellhomogenates, disrupted cells, etc. and can be prepared in a variety ofways. For example, a paste of frozen dead cells may be prepared, livingcells may be frozen or living cells may be used directly in the methodof the current invention.

The method of the current invention relates to filtering suspendedparticles from soluble protein solutions. Suspended particles arefrequently formed when cells are lysed and may include insolubleprecipitates along with cell debris. Further, suspended particles areoften found in solutions of soluble proteins secreted either bymicroorganisms or eucaryotes. Suspended particles are often difficult toremove from soluble protein solutions because of their small size.

In one embodiment, suspended particles are removed from a solubleprotein solution obtained by secretion. In another embodiment, suspendedparticles are removed from a soluble protein solution obtained by celllysis.

In a preferred embodiment, bacterial cells are lysed to form solubleprotein solutions. In a specific embodiment, E. coli cells that expressa heterologous protein, such as SY161, are lysed. In another embodiment,E. coli cells that express wild type protein are lysed.

A number of methods well-known in the art may be used to lyse bacterialcells such as bead mills, osmotic shock, freeze fracture and enzymatictreatment. Preferably, a high pressure homogenizer, such as amicrofluidizer, is used to lyse bacterial cells.

Lysis of bacterial cells releases substantial amounts of DNA andendotoxins into the lysate. Preferably, the amount of DNA and endotoxinsin the bacterial lysate are reduced prior to removing suspendedparticles. Many methods for removing DNA and endotoxins from bacteriallysates are known to those of skill in the art. These methods, includebut are not limited to, ammonium sulfate precipitation, anion exchangechromatography or filtration.

In a preferred embodiment, DNA and endotoxins in a bacterial lysate arereduced by flocculation. Preferably, flocculation is performed withpolyethyleneimine at between about pH 7.3 and about pH 7.7. Otherflocculation methods are known to those of skill in the art. Typically,flocculation reduces the amount of DNA in the lysate by between about100-fold to about 150-fold and the amount of endotoxins by between about1,000-fold and about 10,000-fold, as measured by conventional DNAthreshold methods and Limulus Amoebocyte Lysate (LAL) methods,respectively.

Flocculant and suspended particles are then removed from lysate byfiltration through highly purified diatomaceous earth. Diatomaceousearth (i.e., kieselguhr), is a light colored, porous sedimentary rockcomposed of the silaceous shells of diatoms, which are unicellar aquaticplants of microscopic size. When well hardened, diatomaceous earth iscommonly called diatormite.

Diatomaceous earth has been used in a number of different situations,including but not limited to, separation, adsorption, support andfunctional filler applications (Breese, (1994) “Industrial Minerals andRocks,” 6^(th) ed., Littleton, Colo.: Society for Mining, Metallurgy andExploration, pp. 397-412). Diatomaceous earth is known in the art as afiltration aid and has been used, for example, in the processing ofoils, beverages, solvents and chemicals on an industrial scale.

Diatomaceous earth may be obtained in a variety of different grades andpurity. A significant problem with the use of diatomaceous earthtypically available from commercial suppliers, in biologicalapplications, is leaching of significant amounts of impurities from thediatomaceous earth into biological solutions.

Preferably, highly purified diatomaceous earth is used to practice thecurrent invention. Methods for preparing highly purified diatomaceousearth have been described in the art (Shiuh et al., U.S. Pat. No.5,656,568, which is herein incorporated by reference). Preferably,Celpure™ (most preferably, Celpure™ P-1000) a grade of highly purifieddiatomaceous earth is used to practice the current invention (AdvancedMinerals, Inc., 130 Castilian Drive, Santa Barbara, Calif., 93117).

Highly purified diatomaceous earth such as Celpure™ has the followinggeneral characteristics: extremely high purity, low density, low solubleimpurity content, low total impurity content, and high brightness (Shiuhet al., U.S. Pat. No. 5,656,568). The highly purified diatomaceous earth(i.e., Celpure™) used to practice the invention should have been leachedin appropriate media (e.g., by acid treatment) to remove solubleimpurities, have a total SiO₂ content of at least about 95% and a silicaspecific volume of greater than about 3.4 (Shiuh et al., U.S. Pat. No.5,656,568).

The bead size of the highly purified diatomaceous earth used to practicethe current invention is typically determined by the volume of thesoluble protein solution. While small bead sizes may provide reasonableprotein recovery, the amount of back pressure generated is unacceptablewhen large volumes of soluble protein are filtered. Generally, largerbeads provide a better filter cake and lower back pressure and arepreferred for at least these reasons.

Preferably, the lysate is stirred with highly purified diatomaceousearth before filtration through a filter press. Typically, the yield ofsoluble protein solution after filtration through highly purifieddiatomaceous earth is between about 95% and about 100%, as measured byquantitative reverse phase HPLC.

In some situations, protection of cysteine residues in the solubleprotein solution (preferably, a bacterial lysate containing aheterologous protein expressed in bacteria) may be desirable. Cysteineprotection may prevent protein intermolecular or intramoleculardisulfide bond formation and/or undesirable sulfhydryl oxidation.Preferably, the soluble protein solution is treated with a sulfhydrylprotecting group, which may be selected from the many reagents that havebeen described in the art (see e.g., Greene et al., “Protective Groupsin Organic Synthesis”, Chapter 6, John Wiley & Sons). Appropriatesulfhydryl protecting groups include, but are not limited to,disulfides, sulfenyl compounds, thiocarbamates, thiocarbonates,thioesters, thioethers, etc.

In an exemplary embodiment, the sulfhydryl groups of cysteine residuesof the soluble protein solution (preferably, a bacterial lysatecontaining a heterologous protein) are blocked by oxidation to adisulfide or sulfenyl group. Preferably, sulfonation with sodium sulfateand sodium tetrathionate is used to block the sulfhydryl-groups. Othermethods for forming sulfonates are known to the skilled artisan.Ideally, about a 2:1 ratio of sodium sulfite and sodium tetrathionateare added to the soluble protein solution, which is adjusted to a pH ofbetween about 7.8 and about 8.2. Preferably, when the soluble proteinsolution is a bacterial lysate containing a heterologous protein, thesulfhydryl groups of cysteine residues are protected after cell lysisand before flocculation.

The cysteine protecting group should also be readily removable. Manymethods for converting disulfides, sulfenyl compounds, thiocarbamates,thiocarbonates, thioesters, thioethers, etc. to the free thiol have beendescribed in the art (see e.g., Greene et al., “Protective Groups inOrganic Synthesis”, Chapter 6, John Wiley & Sons). In an exemplaryembodiment, when the cysteine residues in the soluble protein solutionhave been protected by sulfonation, they are deblocked with a reducingagent. Many reducing agents are known in the art and include, but arenot limited to, sodium borohydride, mercaptans (e.g., 2-mercaptoethanol,methythioglycoloate, 3-mercapto-1,2-propanediol, 3-mercaptoproprionicacid, dithioerythritol and dithiothreitol), tri-n-butyl phosphine,hydrogen in the presence of noble metal catalysts and alkali in liquidammonia. Preferably, dithiothreitol is used to deprotect the cysteineresidues of the soluble protein solution when a sulfonate has been usedas the protecting group. The cysteine protecting group may be removedafter lysate flocculation (e.g., when the soluble protein solution is abacterial lysate containing a heterologous protein) or any othersubsequent purification step.

In some cases, substantial amounts of heterologous protein may beprecipitated within the bacterial cell as refractile bodies. In thissituation, cell lysis will provide a lysate that contains substantialamounts of refractile bodies. Preferably, these refractile bodies areresolubilized and the resulting heterologous protein restored to activeform, prior to removing suspended particles from the lysate. Otherwise,large quantities of the heterologous protein will be removed duringfiltration, which greatly reduces the overall yield of the process.Methods for resolubilizing refractile bodies and restoring the resultingheterologous protein to active form are known to the skilled artisan(see, e.g., Jones et al., U.S. Pat. No. 4,512,922). Preferably,refractile bodies are resolubilized and restored to active form prior tolysate flocculation.

5.3 Processing of the Soluble Protein Solution Following Removal ofSuspended Particles

Soluble protein solutions may be further processed, for example, inorder to provide a soluble protein solution of a higher level of purity.The level of purity required will depend on the potential use of theprotein. For example, therapeutic uses will typically require extensivefurther purification following application of the method of the currentinvention.

Any protein purification methods known to the skilled artisan may beused for further purification. Such techniques have been extensivelydescribed in “New Protein Techniques: Methods in Molecular Biology,”Walker, J. M., ed., Humana Press, Clifton, N.J., 1988; and ProteinPurification: Principles and Practice, 3rd. Ed., Scopes, R. K.,Springer-Verlag, New York, N.Y., 1987. In general, techniques including,but not limited to, ammonium sulfate precipitation, centrifugation, ionexchange chromatography, affinity chromatography, gel filtration,reverse-phase chromatography (and the HPLC or FPLC forms thereof), andadsorption chromatography may be used to further purify a solubleprotein solution.

The following examples are provided to further illustrate the currentinvention but are not intended to limit the scope of the currentinvention in any way.

6. EXAMPLE 1 Removing Suspended Particles from E. Coli Lysate ContainingSY161

6.1. Lysis of E. Coli Cells Expressing SY161

SY161 may be produced in E. coli strain TG1 transformed with plasmidpMc5-SY161-S3C. This clone represents 13 mutations from the originalStaphylokinase gene subcloned from Staphylococcus aureus.

The E. coli cells expressing SY161 were harvested by centrifugation andstored at −70° C. prior to use. The frozen cell paste was broken intopieces and suspended in about 7.0 volumes (weight/volume) of lysatebuffer (50 mM sodium phosphate, pH 9.5 containing 5 mM EDTA) using, anoverhead mixer set at between about 500 RPM to about 1000 RPM. Mixingwas continued until the cell paste was completely suspended in thelysate buffer. A microfluidizer unit was assembled by connecting therequired air pressure lines, coolant lines and hoses. The microfluidizerwas then purged with lysate buffer and the pressure was adjusted tobetween about 13,000 psi to about 14,000 psi. The suspended cell pastewas transferred to a pressure vessel, which was then sealed and adjustedto a pressure of about 30 psi. A stainless steel in-line filter was thenconnected to the bottom of the pressure vessel to prevent large cellclumps from clogging the microfluidizer. A feed line was attached to thepressure vessel containing the suspended cell paste. The homogenizer wasturned on, the feed valve was opened and the pressure of the system wasallowed to equilibrate until it was between about 13,000 psi to about14,000 psi. The once-lysed cell suspension was collected in a clean tankand the system was rinsed with appropriate quantities of lysate buffer.The above procedure was then repeated to provide a twice-lysed cellsuspension containing SY161.

6.2. Lysate Sulfonation

The lysate prepared in Section 5.1 was stirred until well suspended. Ifnecessary, the pH of the lysate was adjusted to about 8.0±0.2 witheither dilute acid or base. The target lysate volume may be calculatedby multiplying the weight of the cell paste used in the procedure aboveby 10. Lysate buffer was added with stirring until the desired volumewas reached. The amount of sulfitolysis stock solution that was added tothe lysate may be readily approximated by multiplying the lysate volumeby 0.05 (the stock solution was a mixture of 200 mg/ml sodium sulfiteand 100 mg/ml sodium tetrathionate). The appropriate amount ofsulfitolysis stock solution was added to the lysate, which was mixed forabout 4.0 hours at room temperature until sulfonation of SY161 wascomplete.

6.3. Lysate Flocculation

10% phosphoric acid was slowly added with mixing to the sulfonatedlysate prepared in Section 5.2 until the lysate reached a pH of about7.5±0.2. A 5% (w/w) polyethyleneimine (“PEI”) stock solution wasprepared by diluting 50% PEI to 5% and adjusting the pH to about 7.5±0.2with hydrochloric acid. The volume of PEI stock solution used forflocculation may be estimated by dividing the volume of sulfonatedlysate by 25. The appropriate amount of PEI stock solution was added tothe lysate to provide a final PEI concentration of about 0.2%. The flowrate of PEI addition was a critical parameter and may be calculated bymultiplying the volume of pH adjusted sulfonated lysate by 0.8, whichprovided an appropriate flow rate in milliliter per minute. If PEI wasadded at too rapid of a rate the product protein was co-flocculated,which significantly reduced the yield of the process. The calculatedvolume of 5% PEI was added to the sulfonated lysate at the flow ratecalculated from the formula provided above. The sulfonated lysate wasgently stirred during PEI addition, although vortexing or foaming wasavoided.

6.4. Lysate Filtration

The amount of highly purified diatomaceous earth (e.g., Celpure™ P-1000)added to the lysate prepared in Section 5.3 may be estimated bymultiplying the volume of the lysate in liters by 0.06, which provided aCelpure™ P-1000 concentration of about 6%. Table 1 provides therelationship among the bead size, percentage of activity and productrecovery. Small beads such as Celpure™ P-65 provided reasonable recoverybut generated higher back-pressure, which is unacceptable in largescale. Generally, larger beads provided a better filter cake andpreferred for this reason. The calculated amount of Celpure™ P-1000 wasadded to the lysate with mixing. Importantly, mixing should be done atthe lowest rate necessary to provide a suspension of Celpure™ P-1000.TABLE 1 Test Activity (HU/Assay) Recovery (%) Total Lysate (%) 43.46 1004% Celpure ™ P-65 37.51 86.31 6% Celpure ™ P-65 27.85 64.09 2% Celpure ™P-300 36.74 84.54 4% Celpure ™ P-300 33.11 76.18 6% Celpure ™ P-30040.62 93.47 4% Celpure ™ P-1000 35.48 81.63 6% Celpure ™ P-1000 48.52111.65

The filter press was prepared as follows. Fresh filter pads (preferably,filter cloth septums from Nylon filter cloth S/46412-4-CHS made byKomline-Sanderson) were installed and a filter press (preferably, aBegerow BECO-ASF 4000 filter press) was rinsed and equilibrated. Itshould be noted that Nylon filter cloth was critical to filtration ofthe lysate through highly purified diatomaceous earth. The hold-upvolume of the filter press may be estimated at this time. The amount ofCelpure™ P-1000 pre-coat used in the filter press may be calculated bymultiplying the filtration surface area, in square meters, by 1000 (eachfilter sheet is 0.14 m²). The required amount of Celpure™ P-1000 wassuspended in approximately 50 liters of filtration buffer (i.e., 50 mMsodium phosphate, pH 7.5) and the filter press was pre-coated bycirculating the Celpure™ P-1000 suspension through the filter pressuntil the suspension became clear. Lysate filtration commencedimmediately at a flow rate of between about 5 and about 10 liters perminute. The filtrate and filter cake wash were collected and any liquidremaining in the filter press was removed by flushing with compressedair. The filter cake retained cell debris such as suspended particlesand flocculated material. The turbidity of the lysate was reduced fromabout 1800 Nephelometric Turbidity Units (“NTU”) to less than about 10NTU. The yield of SY161 was between about 95% and about 100%. The clearsolution was directly used in further applications.

7. EXAMPLE 2 Removing Suspended Particles from E. Coli Lysate

7.1 Lysis of E. Coli Cells that do not Express a Heterologous Protein

E. coli null cells (E. coli TG1, pMc5-8 (Δ clone)) for expression ofSY161 were harvested by centrifugation and stored at −70° C. prior touse. The frozen cell paste was suspended in about 7.0 volumes(weight/volume) of lysate buffer (50 mM sodium phosphate buffer, pH 9.5,containing 5 mM EDTA). The frozen cell paste was stirred for about 0.5hour with a Silverson Lab Mixer Emulsifier (Model L4R) at about 3,000rpm to resuspend the cells. A microfluidizer (Model 110Y) was connectedto compressed air and the cooling chamber was filled with ice. Thehomogenizer was purged with lysate buffer and the pressure was adjustedto between about 13,000 psi to about 14,000 psi. The suspended cellswere fed into a homogenizer and lysed under the operational pressure ofbetween about 13,000 psi to about 14,000 psi; The once-lysed cellsuspension was collected in a clean container and the system was rinsedwith appropriate quantities of lysate buffer. The above procedure wasthen repeated to provide a twice-lysed cell suspension containing E.coli host cell proteins.

7.2 Lysate Flocculation

10% phosphoric acid was slowly added to the lysate prepared in Section7.1 with mixing, until the lysate pH reached a pH of about 7.5±0.2. A 5%(w/w) PEI stock solution was prepared by diluting 50% PEI to 5% andadjusting the pH to 7.5±0.2 with hydrochloric acid. The appropriateamount of PEI stock solution was then added to provide a final PEIconcentration of about 0.2%. The flow rate of PEI addition wascalculated by multiplying the volume of the lysate by 0.8, whichprovided an appropriate flow rate in milliliter per minute. If PEI wasadded too rapidly, the E. coli proteins can be co-flocculated whichwould significantly reduce the yield of the process. The lysate wasgently stirred during the PEI addition, although vortexing or foamingwas avoided.

7.3 Lysate Filtration

The amount of highly purified diatomaceous earth (e.g., Celpure™ P-1000)added to the flocculated lysate was estimated by multiplying the volumeof the flocculated lysate in liters by 0.09, which provides a Celpure™P-1000 concentration of 9%. The calculated amount of Celpure™ P-1000(about 450 g of Celpure™ P-1000 was added to about 5 L of lysate) wasadded to the lysate with mixing.

A Komline-Sanderson Avery Filter Press, Model 177 Laboratory FilterPress and Nylon filter cloth were used in the filtration process. Thesystem hold-up volume was about 1.8 L. The lysate-DE mixture wasrecycled by pumping the mixture through the filter press with aSandpiper pump, (model PB ½-A) and maintaining the air pressure betweenabout 25 psi and 40 psi until the filtrate was cleared. The filtrate wasthen directed with an outlet tube to a clean container. The hold-upliquid was removed by connecting the filter press to compressed air. AnSDS-PAGE gel indicated that no protein species were lost duringflocculation and filtration. However, most DNA and endotoxin wereremoved from the lysate.

The addition of PEI was critical for removing suspended particles fromthe soluble protein solution as shown in Table 2. Table 2 illustratesendotoxin removal and removal of suspended particles from lysate withand without PEI addition.

Removal of suspended particles in the lysate was conveniently monitoredby turbidity measurements of the lysate using an HACH 2100 ANturbidimeter. Endotoxin removal was measured by LAL. TABLE 2 Comparisonof an E. coli lysate after PEI addition and filtration with an E. colilysate after filtration without PEI addition. Turbidity (NTU) EndotoxinTotal Endotoxin Unit Line Exp Sample Dilution Reading NTU per ml (EU/ml)1 1 Lysate 15 125 1875 >3,000,000 2 1 Lysate after 1 7 7 30 PEI additionand filtration 3 2 Lysate 15 175 2625 >3,000,000 4 2 Lysate after 1 130130 3,000,000 filtration.

The turbidity reading of the lysate was 1875 NTU, while the amount ofendotoxins was greater than about 3,000,000 EU/ml (see line 1, Table 2)prior to addition of PEI and filtration in Experiment 1. Afterfiltration, the amount of endotoxins (i.e., 30 EU) and suspendedparticles (i.e., 7 NTU) were greatly reduced (compare line 1 to line 2,Table 2) in Experiment 1.

The turbidity reading of the lysate was 2625 NTU, while the amount ofendotoxins were greater than about 3,000,000 EU/ml (see line 3, Table 3)prior to filtration in Experiment 2 (no PEI added). After filtration,the amount of endotoxins (i.e., 3,000,000 EU) and suspended particles(i.e., 130 NTU) were reduced (compare line 3 to line 4, Table 2) butwere significantly greater than when PEI was added to the lysate inExperiment 1 (compare line 2 and line 4, Table 2). Thus, PEI additionmay increase the removal of suspended particles by about 10-20-fold andreduce the amount of endotoxin in the filtrate by about 10,000-fold.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention and any sequences which are functionally equivalent are withinthe scope of the invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

1-23. (canceled)
 24. A method for removing suspended particles from asoluble protein solution comprising the step of filtering the solubleprotein solution through highly purified diatomaceous earth, therebyproviding a clarified soluble protein solution.
 25. The method of claim24, wherein the soluble protein solution is a secreted protein solution.26. The method of claim 25, wherein the soluble protein solution is alysate.
 27. The method of claim 26, wherein the lysate is a bacteriallysate.
 28. The method of claim 26, wherein the lysate is a bacteriallysate containing a heterologous protein that was obtained by expressionin bacteria.
 29. The method of claim 24, further comprising the step ofstirring the soluble protein solution with a highly purifieddiatomaceous earth before filtering through a filter press.
 30. Themethod of claim 24, wherein the yield of the soluble protein solution isbetween about 95% and about 100%.
 31. The method of claim 24, whereinthe highly purified diatomaceous earth is CELPURE.
 32. The method ofclaim 27, wherein the bacteria is E. coli.
 33. The method of claim 28,further comprising the step of blocking cysteine residues of theheterologous protein.
 34. The method of claim 33, wherein the cysteineresidues are blocked with an oxidizing agent.
 35. The method of claim34, wherein the oxidizing agent comprises sodium sulfite.
 36. The methodof claim 34, wherein the oxidizing agent comprises sodium tetrathionate.37. The method of claim 34, wherein the oxidizing agent is a 2:1 ratiomixture of sodium sulfite and sodium tetrathionate.
 38. The method ofclaim 34, wherein the oxidizing agent is added to the protein solutionat a pH of between about 7.8 and about 8.2.
 39. The method of claim 33,further comprising the step of deblocking the blocked cysteine residues.40. The method of claim 39, wherein the blocked cysteine residues aredeblocked with a reducing agent.
 41. The method of claim 39, wherein theblocked cysteine residues are deblocked with dithiothreitol.
 42. Themethod of claim 28, further comprising resolubilizing refractile bodiesin the lysate.
 43. The method of claim 28, in which the heterologousprotein is SY161, wherein SY161 has an amino acid sequence as shown inSEQ ID NO 1.